1. Field of the Invention
This invention pertains to the field of enzymatic synthesis of oligosaccharides using fusion proteins that can catalyze more than one reaction involved in the enzymatic synthesis.
2. Background
Increased understanding of the role of carbohydrates as recognition elements on the surface of cells has led to increased interest in the production of carbohydrate molecules of defined structure. For instance, compounds comprising the sialyl Lewis ligands, sialyl Lewisx and sialyl Lewisa are present in leukocyte and non-leukocyte cell lines that bind to receptors such as the ELAM-1 and GMP 140 receptors. Polley et al., Proc. Natl. Acad. Sci. USA (1991) 88: 6224 and Phillips et al. (1990) Science 250: 1130, see, also, U.S. Pat. No. 5,753,631.
Because of interest in making desired carbohydrate structures, glycosyltransferases and their role in enzyme-catalyzed synthesis of carbohydrates are presently being extensively studied. These enzymes exhibit high specificity and are useful in forming carbohydrate structures of defined sequence. Consequently, glycosyltransferases are increasingly used as enzymatic catalysts in synthesis of a number of carbohydrates used for therapeutic and other purposes. In the application of enzymes to the field of synthetic carbohydrate chemistry, the use of glycosyltransferases for enzymatic synthesis of carbohydrate offers advantages over chemical methods due to the virtually complete stereoselectivity and linkage specificity offered by the enzymes (Ito et al. (1993) Pure Appl. Chem. 65: 753; and U.S. Pat. Nos. 5,352,670, and 5,374,541).
Chemoenzymatic syntheses of oligosaccharides and of corresponding derivatives therefore represent an interesting opportunity to develop novel therapeutic agents. However this approach is still hampered by the relatively poor availability of the required glycosyltransferases and the difficulty and cost of obtaining substrates for these enzymes. Large-scale enzymatic syntheses of oligosaccharides will also require large amounts of the accessory enzymes necessary for the synthesis of the sugar-nucleotides that are used as the donors by the glycosyltransferases. The present invention provides fusion proteins that simplify the purification of enzymes that are useful for enzymatic synthesis of oligosaccharides.